Purification and characterization of two types of NADH-quinone reductase from Thermus thermophilus HB-8

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Abstract
Two types of the NADH-quinone reductase were isolated from Thermus thermophilus HB-8 membranes, by use of the nonionic detergent, dodecyl .beta.-maltoside, and NAD-agarose affinity, DEAE-cellulose, hydroxyapatite, and Superose 6 column chromatography. One of these (NADH dehydrogenase 1) is a complex composed of 10 unlike polypeptides, and the other (NADH dehydrogenase 2) exhibits a single band (Mr 53,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 1 was about 14 times higher than that of the dodecyl .beta.-maltoside extract and partially rotenone sensitive. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 2 was about 30-fold as high as that of the dodecyl .beta.-maltoside extract and rotenone insensitive. The purified NADH dehydrogenase 1 contained noncovalently bond FMN, non-heme iron, and acid-labile sulfide. The ratio of FMN to non-heme iron to acid-labile sulfide was 1:11-12:7-9. The high content of iron and labile sulfide is suggestive of the presence of several iron-sulfur clusters. The purified NADH dehydrogenase 2 contained noncovalently bound FAD and no non-heme iron or acid-bile sulfide. The activities of both NADH dehydrogenases were stable at temperatures of .gtoreq. 80.degree. C. The occurrence of two distinct types of NADH dehydrogenase as a common feature in the membranes of various aerobic bacteria is discussed.